【生物】第四章--突变蛋白质物理化学性质分析.ppt

第四章第四章:突变蛋白质的突变蛋白质的物理化学性质分析物理化学性质分析第一节蛋白质溶液的热力学第二节蛋白质折叠动力学第三节突变,稳定性和折叠第一节蛋白质溶液的热力学第一节蛋白质溶液的热力学n nI.I.热运动与蛋白质构象热运动与蛋白质构象 “生命的还原论生命的还原论”摆动摆动,振动振动,转动转动-多肽折叠过程所涉及的主要运动形式多肽折叠过程所涉及的主要运动形式;热力学平衡热力学平衡;n nII.II.热力学函数与热力学平衡热力学函数与热力学平衡n nIII.III.热容量热容量:某物质的热容量是某物质的比热与该物质质量的乘积。即某物质的热容量是某物质的比热与该物质质量的乘积。即Cm.Cm.n n单位质量的某种物质温度升高单位质量的某种物质温度升高1 1吸收的热量叫做吸收的热量叫做这种物质的比热容，简称：比热，用字母这种物质的比热容，简称：比热，用字母“c”c”表表示。示。n n摩尔热容量的定义，即摩尔热容量的定义，即1 1摩尔物质温度升高（或降低）摩尔物质温度升高（或降低）1 1度时所吸收度时所吸收（或放出）的热量，用（或放出）的热量，用C C表示，单位是表示，单位是J/J/（molKmolK）.比热的单位为比热的单位为 cal/cal/n nIV.vant Hoff IV.vant Hoff 焓焓n nV.V.折叠折叠/退折叠转变退折叠转变 “协同与独立协同与独立”n nVI.VI.量热法与折叠过程热力学量热法与折叠过程热力学第二节蛋白质折叠动理学第二节蛋白质折叠动理学n nI.折叠动理研究技术:FLOW;Stopped-flow 光化学触发;温度或压力突变;超快混合技术.n nII.两态动理n nIII.过度态n n1)折叠过程与过度态n n2)对折叠过度态的性质分析第二节蛋白质折叠动理学第二节蛋白质折叠动理学(续续1)n nIV.折叠的中间态n n1).熔球态n n2).快态与慢态n n3).二硫键引起的中间态n n4).多结构域蛋白的折叠第二节蛋白质折叠动理学第二节蛋白质折叠动理学(续续2)n nV.折叠的基本过程n n1).接触形成n n2).螺旋-链环转变n n3).b b 发卡形成第三节突变第三节突变,稳定性和折叠稳定性和折叠n nI.热力学参数在分子水平上的解释n n1).静电相互作用n n2).范德华相互作用n n3).氢键n n4).疏水效应n n5).二硫键第三节突变第三节突变,稳定性和折叠稳定性和折叠(续续1)n nII.突变与热稳定性n n1).疏水突变n n2).氢键突变n n3).适应极端条件的突变体第三节突变第三节突变,稳定性和折叠稳定性和折叠(续续2)n nIII.突变与折叠过程n n1).过渡态的突变分析n n2).突变对稳定性和折叠过程的影响How do proteins fold into tertiary structure?How do proteins fold into tertiary structure?n nQuickly most single domains fold on millisecond timescalesQuickly most single domains fold on millisecond timescalesn nHighly cooperative specific folding intermediates are rarely observedHighly cooperative specific folding intermediates are rarely observedn nTraditional view:ITraditional view:I II II III III IV describes order of structure formation IV describes order of structure formationn nModern view:local nucleation of II structure elements combined with Modern view:local nucleation of II structure elements combined with condensation or collapse of hydrophobic groupscondensation or collapse of hydrophobic groupsn nAlternative perspectives-specific folding pathways vs.energy landscapes that Alternative perspectives-specific folding pathways vs.energy landscapes that flow downhill from initial unfolded to final native structureflow downhill from initial unfolded to final native structuren nCotranslationallyCotranslationally?modular proteins(common in eukaryotes,not in prokaryotes)?modular proteins(common in eukaryotes,not in prokaryotes)may require this(but protein may require this(but protein synthsynth in bacteria is 10 x faster than in eukaryotes-in bacteria is 10 x faster than in eukaryotes-so this may define extent of so this may define extent of cotranslationalcotranslational folding)folding)n nWhat are driving forces?Entropic or What are driving forces?Entropic or enthalpicenthalpic?Forces that stabilize native?Forces that stabilize native structure are not necessarily those driving the formation of the structurestructure are not necessarily those driving the formation of the structuren nIn aqueous(polar)environment,H-bond to HIn aqueous(polar)environment,H-bond to H2 2O is equivalent to H-bond between two O is equivalent to H-bond between two components of polypeptide chain:Hcomponents of polypeptide chain:H2 2O-H-N O-H-NC=O-H-NC=O-H-Nn nThus,no stabilization from II structure formation H-bondingThus,no stabilization from II structure formation H-bondingn nBut in buried hydrophobic(But in buried hydrophobic(apolarapolar)environment,H-bond may be more)environment,H-bond may be more enthalpicallyenthalpically favoredfavoredn nStudying protein folding process is difficult-mixtures,not single molecules;few Studying protein folding process is difficult-mixtures,not single molecules;few measurements can work at required timescales;use perturbations to disrupt measurements can work at required timescales;use perturbations to disrupt native structure(T,pH,denaturant,pressure),monitor refolding kineticsnative structure(T,pH,denaturant,pressure),monitor refolding kineticsn nSome proteins Some proteins misfoldmisfold if they dont have help-chaperones if they dont have help-chaperonesProtein folding/unfoldingProtein folding/unfoldingn nAnfinsenAnfinsen showed that proteins showed that proteins(RNaseRNase)spontaneously refold to)spontaneously refold to the same unique three-the same unique three-dimensional structure,dimensional structure,therefore,amino acid sequence therefore,amino acid sequence alone encodes tertiary structure alone encodes tertiary structure(Nobel prize,1972)(Nobel prize,1972)n nC.B.C.B.AnfinsenAnfinsen,Science(1973),Science(1973)181,223-239.181,223-239.n nNative configurations of folded Native configurations of folded proteins are marginally stableproteins are marginally stablen nLarge stabilizing and Large stabilizing and destabilizing factors are destabilizing factors are balanced,resulting in balanced,resulting in folded state that can be folded state that can be easily disrupted by elevated easily disrupted by elevated temperature,pressure or temperature,pressure or chemical denaturantschemical denaturantsProtein folding/unfoldingProtein folding/unfoldingn nWhat are the forces/factors involved in stabilizing protein structure?What are the forces/factors involved in stabilizing protein structure?n nGibbs free energy of folding is negative(folded protein is stable)but small Gibbs free energy of folding is negative(folded protein is stable)but small(folded state is easily perturbed)(folded state is easily perturbed)D D D DG=G=D D D DH TH TD D D DS 0 for S 0,D DG0)Models for protein foldingModels for protein foldingn nHow does folding occur?Still an How does folding occur?Still an active area of researchactive area of researchn nFolding cannot occur randomlyFolding cannot occur randomly LevinthalsLevinthals paradox:not enough time paradox:not enough time to sample all possible conformations,to sample all possible conformations,therefore must follow specific folding therefore must follow specific folding pathways pathways C.C.LevinthalLevinthal,J.,J.ChimChim.Phys.(1968).Phys.(1968)65,44-45.65,44-45.n nLed to hierarchical,backbone/2 Led to hierarchical,backbone/2 structure-centric viewstructure-centric viewn nfirst form secondary structure first form secondary structure elements(stabilized by H-bonding),elements(stabilized by H-bonding),n nthen assemble into folded domains then assemble into folded domains(stabilized by hydrophobic(stabilized by hydrophobic interactions)interactions)n nLater,sidechain-centric view Later,sidechain-centric view assumed folding driven by assumed folding driven by hydrophobic collapse-loosely hydrophobic collapse-loosely assembled core interactions drive assembled core interactions drive formation of compact 2 structuresformation of compact 2 structuresn nCurrent debate works to incorporate Current debate works to incorporate roles of 2 structure and collapse into roles of 2 structure and collapse into single model:nucleation-single model:nucleation-condensationcondensationn nBut does a given protein follow the But does a given protein follow the same pathway,or can multiple same pathway,or can multiple pathways(reaction coordinates)yield pathways(reaction coordinates)yield same final structure?same final structure?Models for protein foldingModels for protein foldingn nTwo views of folding-micro Two views of folding-micro vsvs macro macron nHierarchical view assumes Hierarchical view assumes specific sequence of events specific sequence of events(paths,intermediates,(paths,intermediates,transition states)for transition states)for individual molecules individual molecules(sequential(sequential micropathmicropath view)view)n nEnsemble view considers Ensemble view considers heterogeneity of the starting heterogeneity of the starting pointpointn nunfolded state of the unfolded state of the ensemble is not ensemble is not homogeneous homogeneous n neach unfolded protein each unfolded protein molecule has a different molecule has a different conformationconformationn nFunnel explains fast folding Funnel explains fast folding through general description through general description of energy landscapeof energy landscapen nunfolded proteins roll unfolded proteins roll downhill toward smaller downhill toward smaller populations of populations of conformationsconformationsn nfolding intermediates reflect folding intermediates reflect kinetic traps-local minima kinetic traps-local minima on surface of landscapeon surface of landscapen neach protein follows a each protein follows a different pathway to native different pathway to native structure(by definition,structure(by definition,since each has a since each has a uniqeuniqe starting point)starting point)Protein folding landscapesProtein folding landscapesn nFunnel shape represents number of Funnel shape represents number of equivalent states at a given free energy equivalent states at a given free energy level(many unfolded;one native)level(many unfolded;one native)n nSmooth landscapes represent fast,two-Smooth landscapes represent fast,two-state folding;increased ruggedness state folding;increased ruggedness corresponds to slower folding with corresponds to slower folding with intermediates(kinetic traps;local minima)intermediates(kinetic traps;local minima)n nMore research needed to characterize More research needed to characterize energy landscapes experimentallyenergy landscapes experimentallyProtein folding problemProtein folding problemn nAnfinsenAnfinsen showed amino acid sequence alone encodes tertiary structure showed amino acid sequence alone encodes tertiary structuren nHence the Protein folding problem-it should be possible to predict 3D Hence the Protein folding problem-it should be possible to predict 3D protein structures from genetic information-protein structures from genetic information-as yet unsolvedas yet unsolvedn nKen Dill,Michael Ken Dill,Michael LevittLevitt and others use simplified amino acid alphabets(H,P)and others use simplified amino acid alphabets(H,P)and 2D models to simulate folding and test hypotheses based upon and 2D models to simulate folding and test hypotheses based upon energy energy landscapeslandscapes and hydrophobic collapse to predict protein structures from and hydrophobic collapse to predict protein structures from sequence alonesequence alone (For review,see Dill,K.Protein Science(1999),8:1166-1180.)(For review,see Dill,K.Protein Science(1999),8:1166-1180.)n nCASP competitions pair protein structure prediction efforts up with unknown CASP competitions pair protein structure prediction efforts up with unknown proteins currently being determined experimentally to enable real-world proteins currently being determined experimentally to enable real-world testing of prediction methods:testing of prediction methods:http:/http:/predictioncenter.llnl.govpredictioncenter.llnl.gov/n nOthers use various semi-empirical strategies to generate fold predictions-Others use various semi-empirical strategies to generate fold predictions-homology modeling homology modeling n nSwiss-model server for automated homology modeling:Swiss-model server for automated homology modeling:http:/swissmodel.expasy.org/http:/swissmodel.expasy.org/n nSan Diego Supercomputing Center modeling server:San Diego Supercomputing Center modeling server:http:/cl.sdsc.edu/hm.htmlhttp:/cl.sdsc.edu/hm.htmln nHowever,some proteins require help to fold properly:However,some proteins require help to fold properly:CHAPERONINSCHAPERONINSChaperone-assisted foldingChaperone-assisted foldingn nMolecular chaperones-proteins that bind nonnative states of other proteinsMolecular chaperones-proteins that bind nonnative states of other proteinsn noriginally identified at heat-shock proteins(originally identified at heat-shock proteins(HspsHsps),needed to help refold proteins denatured in),needed to help refold proteins denatured in cytosolcytosoln nchaperones prevent aggregation by binding to hydrophobic regions of unfolded/partially folded proteinschaperones prevent aggregation by binding to hydrophobic regions of unfolded/partially folded proteinsn n3 classes of proteins in vivo:spontaneous folding(unassisted);Hsp70 dependent;3 classes of proteins in vivo:spontaneous folding(unassisted);Hsp70 dependent;GroELGroEL dependentdependentn nProbably all proteins interact with trigger factor(TF-bacteria)or nascent chain-associated Probably all proteins interact with trigger factor(TF-bacteria)or nascent chain-associated complex(NAC-eukaryotic)when being translated-ribosomal chaperonescomplex(NAC-eukaryotic)when being translated-ribosomal chaperonesprokaryoteseukaryotesGetting Newly Synthesized Proteins into Shape Bernd Bukau,Elke Deuerling,Christine Pfund,and Elizabeth A.Craig,Cell,(2000)101,119122.http:/ foldingHsp70/Hsp40-mediated foldingn nDnaKDnaK(Hsp70)binds hydrophobic patches of polypeptide as it exits the (Hsp70)binds hydrophobic patches of polypeptide as it exits the ribosome tunnel;prevents aggregation,ribosome tunnel;prevents aggregation,misfoldingmisfolding(passive)(passive)n nBinding of Binding of DnaJDnaJ(Hsp40)+ATP hydrolysis produces conformational change in (Hsp40)+ATP hydrolysis produces conformational change in DnaKDnaK,followed by,followed by GrpEGrpE binding(active)binding(active)n nMultiple rounds of Hsp70 binding/release may occur on a single polypeptideMultiple rounds of Hsp70 binding/release may occur on a single polypeptideGroELGroEL-mediated Folding-mediated Foldingn nRings of Rings of GroELGroEL subunits enclose two cavities for protein subunits enclose two cavities for protein binding and foldingbinding and foldingn nActivities of cavities are coupled,protein folding events Activities of cavities are coupled,protein folding events alternate from one side to the otheralternate from one side to the otherGroELGroEL-mediated Folding-mediated Foldingn nOne protein binds to hydrophobic walls of a ring,contacting 3 or more individual One protein binds to hydrophobic walls of a ring,contacting 3 or more individual GroELGroEL subunits subunitsn nATPaseATPase activity in other ring activates conformational change-hydrophobic activity in other ring activates conformational change-hydrophobic patches become hydrophilicpatches become hydrophilicn nProtein dissociates from walls of cavity into center(Protein dissociates from walls of cavity into center(AnfinsenAnfinsen cage)to promote cage)to promote folding;folding;GroESGroES acts as lid acts as lidn nAfter 15s,After 15s,GroELGroEL subunits subunits hydrolysehydrolyse ATP,ATP,GroESGroES dissociates,folded protein is dissociates,folded protein is releasedreleasedOther factors/practical considerationsOther factors/practical considerationsn nDisulfide bondsDisulfide bondsn nwont form in bacterial wont form in bacterial cytosolcytosol,requires oxidation during,requires oxidation during purification processpurification processn nProlineProline ciscis/trans/trans isomerizationisomerizationn ncatalyzed by catalyzed by cyclophilincyclophilinn ncan promote formation of aggregates due to slow exchange can promote formation of aggregates due to slow exchang